Biomedical subjects

F G Walz

Publications and source records attributed to F G Walz.

At least 19 recordsLinked to original sources

PubMed2001

Probing functional perfection in substructures of ribonuclease T1: double combinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop.

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.

Amino Acid Substitution↗

PubMed1999

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Aspergillus oryzae↗

PubMed1997

Upstream subsite interactions for oligonucleotide binding with ribonuclease T1.

Ribonuclease T1 (RNase T1) specifically cleaves RNA at guanylyl residues. Previous studies revealed the presence of an enzyme-subsite interaction for adenosine residues of ApGpC and ApGpU substrates (Osterman and Walz (1979) Biochemistry 10, 1984-1988). The binding of ApG and 2'-deoxyadenylyl-(3',5')-guanosine (dApG) with RNase T1 was studied in the pH range 5-9 using ultraviolet difference spectroscopy. The association constants for these dinucleoside monophosphates showed the same pH dependence both of which differed at low pH values with that for the methyl phosphoester of 5'-GMP (MepG). This difference suggested that binding of the adenosine group is strongly dependent on the deprotonation of an enzyme/ligand group with a pKa value of < or = 4.8. delta G zero for ApG binding minus that for MepG at pH > 6 yielded a delta delta G of -1.17 +/- 0.10 kcal/mol which is a measure of the contribution of the adenosine moiety to binding. ApG bound more tightly than dApG with a mean delta delta G value of -0.73 +/- 0.10 kcal/mol which demonstrated the involvement of the adenosine 2'-OH group in binding. These and other comparisons indicated that delta delta G for maximal binding the adenine base per se was -0.44 kcal/mol. delta delta G for binding pdApdG minus that for dApdG (-0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.

Adenosine↗

PubMed1996

Adult-male-specific S-warfarin (11S-OH) and progesterone (20 beta-OH) keto-reductases in rat hepatic microsomes are not identical.

Adult-male-specific reductase activities in rat hepatic microsomes use NADPH to reduce S-warfarin and progesterone to their 11S-OH and 20 beta-OH products, respectively (Apanovitch et al. (1992) Biochem. Biophys. Res. Commun. 184, 338-346). When microsomes were treated with increasing concentrations of detergent, S-warfarin (11S-OH) reductase (SW(11S)R) activity was subject to monophasic activation by Triton X-100, monophasic inhibition by sodium cholate, and, activation followed by inhibition with either CHAPS or dodecyl-beta-D-maltoside. A non-dialyzable, heat-sensitive factor in rat and rabbit sera activates microsomal SW(11S)R activity six- to eight-fold. Similar detergent inhibitions but no detergent or serum activations were observed for progesterone (20 beta-OH) reductase (P(20 beta)R) activity. A significant amount of SW(11S)R activity was lost during purification regardless of whether the detergent used for solubilization was activating or inhibiting. Octyl-Sepharose, hydroxyapatite, DEAE-cellulose and carboxymethyl matrices were used to partially purify SW(11S)R. P(20 beta)R activity co-purified with SW(11S)R and the most purified fraction contained two major and several minor polypeptides. Partially purified SW(11S)R is activated by detergents, serum, and salt. These and previous results indicate that SW(11S)R and P(20 beta)R are not identical even though they are both adult male-specific, integral membrane proteins apparently having their active sites exposed on the cytoplasmic surface of the endoplasmic reticulum.

20-Hydroxysteroid Dehydrogenases↗

PubMed1992

Relaxation kinetics of ribonuclease T1 binding with guanosine and 3'-GMP.

Temperature-jump relaxation kinetic studies were undertaken at 25 degrees C with ribonuclease T1 (RNase T1) alone and in the presence of guanosine (Guo) and 3'-guanylic acid (3'-GMP). No relaxations were observed in the absence of ligands and only one process was observed in their presence which reflected a simple on-off reaction in both cases. Apparent association rate constants, k(on), and dissociation rate constants, k(off), were evaluated at several pH values and their ratios, k(on)/k(off), were contrasted with independently determined values of the equilibrium association constant, Ka(eq). The value of k(on)/k(off) for Guo was significantly greater than Ka(eq), whereas Ka(eq) was significantly greater than k(on)/k(off) for 3'-GMP. The simplest interpretation of the result for Guo is that free RNase T1 undergoes a relatively slow undetected isomerization and Guo can bind only with one isomer. 3'-GMP can be considered to bind with the same preference, but in this case the initial enzyme complex undergoes a relatively slow undetected isomerization. These results are consistent with a recent NMR study which suggested that RNase T1 binding with Guo and 3'-GMP are coupled to slow exchange processes in a ligand dependent manner (Shimada, I. and Inagaki, F. (1990) Biochemistry 29, 757-764). It is tentatively concluded that binding of Guo and 3'-GMP at the active site of RNase T1 is limited to a sub-population of conformers involving the base-recognition site and that the phosphomonoester group of the nucleotide can engage in additional conformationally linked interactions at the adjacent catalytic site.

Guanosine↗

PubMed1992

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

20-Hydroxysteroid Dehydrogenases↗

PubMed1992

Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites.

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.

Amino Acid Sequence↗

PubMed1992

Null phenotype for cytochrome P450 2B2 in the rat results from a deletion of its structural gene.

The absence of phenobarbital (PB)-inducible cytochrome P450 2B2 (CYP2B2) in hepatic microsomes from Marshall 520 (M520) and Wistar Munich (WM) inbred strains of rat was previously reported [Biochem. Genet. 25:527-534 (1987)], and it was subsequently shown for M520 rats that corresponding CYP2B2 mRNA was not detected in hepatic extracts from either control or PB-treated animals [DNA 8:29-37 (1989)]. In the present study, solution hybridization was used to quantify PB-induced CYP2B2 and CYP2B1 mRNAs in livers from M520, WM, and outbred Sprague-Dawley rats, as well as additional inbred strains that express all known electrophoretic phenotypes for both of these closely related isozymes. Amounts of these mRNAs were also measured for F1 and F2 progenies of crosses involving M520 rats. The results indicated that the extent of PB induction of both isozymes appears to be independent of the electrophoretic phenotype. It was also shown that the null phenotype for CYP2B2 observed in M520 rats results from a mutation of a single autosomal gene and is inherited codominantly, regarding protein and mRNA phenotypes. Analyses of restriction digests and specific polymerase chain reaction products of hepatic DNAs revealed that the basis for the null phenotype of CYP2B2 in M520 and WM rats was a deletion of the CYP2B2 gene.

Alleles↗

PubMed1990

Spermine stabilization of folded ribonuclease T1.

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form.

Aspartic Acid↗

PubMed1987

Mapping of genes for cytochromes P-450b, P-450e, P-450g, and P-450h in the rat. Demonstration of two gene clusters with P450-b,e localized on chromosome 1.

Inbred ACI, WF, and RCS rats having characteristic markers for albino (c), hemoglobin beta-chain (Hbb), and pink-eyed dilution (p) on chromosome 1 and expressing variants for hepatic cytochromes P-450b, P-450e, P-450g, and P-450h were used in genetic mapping studies for these hemoproteins. The results of WF X (ACI X WF)F1 and RCS X (WF X RCS)F1 backcrosses revealed the existence of two gene clusters designated the P450-b,e and P450-g,h loci. The linkage map P450-b,e-p-c-Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)-p-c-Hbb on mouse chromosome 7. P450-g,h is not linked with P450-b,e and the other markers tested on rat chromosome 1. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 families/subfamilies.

Animals↗

PubMed1987

Polymorphisms of four hepatic cytochromes P-450 in twenty-eight inbred strains of rat.

Two-dimensional gel electrophoresis of hepatic microsomes from phenobarbital-treated animals was used to analyze electrophoretic/regulatory polymorphisms for cytochromes P-450b, P-450e, P-450g, and P-450h in 28 inbred strains of rat. Previous studies with outbred rats revealed the existence of four electrophoretic variants for P-450b, two for P-450e, and three for P-450h as well as two regulatory alleles for P-450g. With the exception of one allozymic form of P-450h, all of these alleles as well as a novel (null) allele for P-450e were found to be homozygous in at least two of the inbred strains tested. Eight phenotypes for combinations of these four cytochromes P-450 were observed. Inbred strains were identified that can be used in studies on the structure/function of unique cytochrome P-450-allozymes and in genetic crosses to map the four distinct cytochrome P-450 genes.

Alleles↗

PubMed1987

A regulatory polymorphism for rat hepatic cytochrome P-450g.

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cis-acting.

Aging↗

PubMed1986

Cytochrome P-450 polypeptides in pulmonary microsomes from rats.

Pulmonary microsomal polypeptides from different strains of rats were resolved using two-dimensional electrophoresis and were further characterized by in situ peptide mapping. Triton X-114 detergent separation was used to enrich cytochromes P-450 (P-450) and other integral membrane proteins from pulmonary microsomes, and these were directly compared with corresponding polypeptides from hepatic microsomes. The results demonstrated that P-450b and epoxide hydrolase were present in the lungs of male and female rats and that their expression in this tissue was independent of phenobarbital treatment. P-450e, which is co-induced with P-450b in the liver, was not detected in pulmonary microsomes under any condition. Four other pulmonary microsomal polypeptides were characterized and preliminary evidence suggested that they represent unique isozymic forms of P-450 with three of them being related to P-450b.

Animals↗

PubMed1985

Microheterogeneity of a male-specific rat hepatic cytochrome P-450: existence of three allozymic forms.

Preparations of hepatic cytochrome P-450 h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were analyzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16 alpha-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P-450 h/2c. Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P-450 h/2c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P-450 h/2c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.

Alleles↗

PubMed1985

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes.

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.

Amino Acid Sequence↗

PubMed1983

Species differences in cytochromes P-450 and epoxide hydrolase: comparisons of xenobiotic-induced hepatic microsomal polypeptides in hamsters and rats.

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.

Animals↗

PubMed1983

At least six forms of extremely homologous cytochromes P-450 in rat liver are encoded at two closely linked genetic loci.

A subpopulation of phenobarbital-induced cytochrome P-450 in rat liver has been shown to consist of four closely related forms of the enzyme that appeared to be strain-related. In the present study, polypeptides composing this family were analyzed by two-dimensional electrophoresis of hepatic microsomes from 64 individual phenobarbital-treated rats. The animals surveyed included both sexes from four inbred and five outbred strains/colonies and F1 progenies from 10 crosses. Two new members of this polypeptide family were identified on the basis of their unique electrophoretic behavior and peptide maps. Eight phenotypes were observed that consisted of two to four member polypeptides. The six closely related cytochromes P-450 were found to be encoded at two genetic loci with at least four alleles at the P-450b locus and at least two alleles at the P-450e locus. Most colonies of outbred strains were characterized by polymorphism at one or both of these loci, and in no case did they contain unique alleles. Analyses of parents and their F1 progenies indicated that the P-450b and P-450e loci are closely linked on the same autosome and are expressed codominantly. Furthermore, the products of these loci appear to be coordinately regulated. The extreme homology between P-450b and P-450e genes, their high degree of polymorphism, and their close linkage suggest that they are subject to the same genetic mechanisms that maintain these features in other multigene families.

Animals↗

PubMed1982

Comparisons of highly purified hepatic microsomal cytochromes P-450 from Holtzman and Long-Evans rats.

The present study describes the purification and characterization of strain variant forms of a major phenobarbital-inducible microsomal hemoprotein, cytochrome P-450b, from Holtzman and Long-Evans rats. The strain variant hemoproteins cannot be resolved by sodium dodecyl sulfate gel electrophoresis, but can be partially separated in two-dimensional isoelectric focusing SDS gels. If, however, sodium tetradecyl sulfate is incorporated into the one-dimensional gel system, separation of the cytochromes P-450b is achieved. Minor structural differences are detected in the peptides of the cytochromes P-450b following limited proteolysis by Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, or reverse-phase high-pressure liquid chromatography of tryptic peptides. The strain variant cytochromes P-450b are immunochemically and spectrally indistinguishable. The optical spectra of the ferric and ferrous hemoproteins are identical, as are the CO- and ethylisocyanide-reduced difference spectra. Ferrous cytochromes P-450b from both rat strains effectively bind metyrapone with equivalent affinities. In addition, the cytochromes P-450b do not differ in their catalytic activities toward benzphetamine, hexobarbital, benzo [a]pyrene, zoxazolamine, 7-ethoxycoumarin, estradiol-17 beta and testosterone. Cytochrome P-450c, the predominant isozyme inducible in rat liver by 3-methylcholanthrene, was purified from Holtzman and Long-Evans rats. Cytochromes P-450c from both rat strains are indistinguishable based on electrophoretic, immunological, spectral and catalytic properties. Minor structural differences in the cytochromes P-450c were revealed in the reverse-phase high-pressure liquid chromatographic profiles of the tryptic peptides of these hemoproteins, but not in the peptides generated by limited proteolysis or cleavage with cyanogen bromide.

Animals↗